Jin Kumagai

INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent

Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4–8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testis Leydig cells and ovarian theca and luteal cells. Male mice mutant for INSL3 exhibit cryptorchidism or defects in testis descent due to abnormal gubernaculum development whereas overexpression of INSL3 induces ovary descent in transgenic females. Because transgenic mice missing the LGR8 gene are also cryptorchid, INSL3 was tested as the ligand for LGR8. Here, we show that treatment with INSL3 stimulated cAMP production in cells expressing recombinant LGR8 but not LGR7. In addition, interactions between INSL3 and LGR8 were demonstrated following ligand receptor cross-linking. Northern blot analysis indicated that the LGR8 transcripts are expressed in gubernaculum whereas treatment of cultured gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation. The present study identified the ligand for an orphan G protein-coupled receptor based on common phenotypes of ligand and receptor null mice. Demonstration of INSL3 as the ligand for LGR8 facilitates understanding of the mechanism of testis descent and allows studies on the role of INSL3 in gonadal and other physiological processes.

Brain-Derived Neurotrophic Factor Promotes Implantation and Subsequent Placental Development by Stimulating Trophoblast Cell Growth and Survival

Successful implantation of the blastocyst and subsequent placental development is essential for reproduction. Expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5, together with their receptor, tyrosine kinase B (TrkB), in trophectoderm cells of blastocyst suggests their potential roles in implantation and placental development. Here we demonstrated that treatment with BDNF promoted blastocyst outgrowth, but not adhesion, in vitro and increased levels of the cell invasion marker matrix metalloproteinase-9 in cultured blastocysts through the phosphatidylinositol 3-kinase pathway. After implantation, BDNF and neurotrophin-4/5 proteins as well as TrkB were expressed in trophoblast cells and placentas during different stages of pregnancy. Both TrkB and its ligands were also expressed in decidual cells. Treatment of cultured trophoblast cells with the TrkB ectodomain, or a Trk receptor inhibitor K252a, suppressed cell growth as reflected by decreased proliferation and increased apoptosis, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using the specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase/Akt pathway in mediating the action of TrkB ligands. In vivo studies in pregnant mice further demonstrated that treatment with K252a, but not K252b, suppressed placental development accompanied by increases in trophoblast cell apoptosis and decreases in placental labyrinth zone at midgestation. In vivo K252a treatment also decreased fetal weight at late gestational stages. Our findings suggested important autocrine/paracrine roles of the BDNF/TrkB signaling system during implantation, subsequent placental development, and fetal growth by increasing trophoblast cell growth and survival.

Survivin acts as an antiapoptotic factor during the development of mouse preimplantation embryos.

Apoptosis is an essential physiologic process used in almost all tissues to remove damaged or superfluous cells. However, the early embryos are unique because no cell death is found up to the blastocyst stage during normal development. Survivin, a member of the IAP family, is capable of binding to caspases to modulate their functions. Here, we investigated the expression of survivin, and its role in preventing apoptosis in mouse preimplantation embryos. Transcripts for survivin and a splice variant lacking exon 2 were detected from unfertilized oocytes up to hatched blastocyst stage. At the protein level, survivin was also detected at all stages of early embryos. The antisense approach was used to demonstrate the role of survivin on embryo development. Development of early embryos treated with antisense survivin oligonucleotides was arrested at the morula or early blastocyst stage with disruption of tubulin formation and abnormal nuclei, associated with apoptosis. The effect of the antisense was enhanced by cotreatment with an apoptosis-inducing reagent, staurosporine. In contrast, apoptosis induced by the antisense treatment was inhibited by caspase-3 and -9 inhibitors. These results indicate that survivin is an essential antiapoptotic gene expressed in preimplantation embryos and could protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspases.

Relaxin signaling in reproductive tissues

The insulin/relaxin peptide family includes insulin, IGFs, relaxin1-3, INSL3/RLF, INSL4, INSL5/RIF2 and INSL6/RIF1, many without functional characterization. Based on analysis of transgenic phenotypes and phylogenetic profiling, we have discovered that two orphan leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8, are cognate receptors for relaxin whereas INSL3 is a specific ligand for LGR8. With the identification of the relaxin receptors, it is now possible to investigate specific cells and tissues that are responsive to relaxin in diverse physiological and pathological conditions as well as to develop agonists and antagonists for LGR7 and LGR8 as therapeutics to treat different labor disorders. Furthermore, future functional characterization of the specificity of these pluripoentent receptors with peptide ligands could lead to the understanding of related orphan ligands and receptors.

The role of leptin during the development of mouse preimplantation embryos

Leptin is known to regulate diverse reproductive functions, and recent studies have implicated involvement of leptin in the early mouse embryo development. The aim of the present study was to investigate the expression of leptin and its functional receptor (OB-Rb) in mouse oocyte and preimplantation embryo, and to examine whether leptin influenced the early embryo development. Leptin mRNA was detected in blastocyst and hatched blastocyst, and OB-Rb mRNA was detected in oocytes, 1-cell, 2-cell, morula, blastocyst and hatched blastocyst. As for the origin of leptin, leptin mRNA was identified in both the oviduct and uterus of the pregnant mouse. Furthermore, in the pregnant mouse, the levels of leptin in uterine fluid were higher than those in the non-pregnant mouse. Supplementation of culture medium with leptin promotes the development of preimplantation embryos from 2-cell stage to the blastocysts, fully expanded blastocysts and hatched blastocysts. Leptin significantly increased the total cell number of blastocysts, and the effect was preferentially observed in the trophectoderm. These findings raise the possibility that leptin regulates the development of mouse preimplantation embryo through a paracrine pathway.

Lack of LGR8 gene mutation in Finnish patients with a family history of cryptorchidism

Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male. Although in Western countries 1-2% of males at the age of 3 months are diagnosed with this condition, its aetiology is still unknown. Animal models suggest a possible genetic basis for this disorder. Recently, the INSL3 (Leydig insulin-like peptide) gene and its cognate receptor, LGR8, were found to be important in testicular descent by regulating gubernacular development. Male mice null for either INSL3 or LGR8 genes exhibited bilateral cryptorchidism. Because earlier studies indicated that mutation of the INSL3 gene is not associated with the development of human cryptorchidism, this study analysed whether mutations in the LGR8 gene could be associated with this disorder. Sequencing of 18 exons of the LGR8 gene in 23 cryptorchid Finnish patients and a group of 33 control subjects allowed the identification of three nucleotide changes in exons 12 and 17, showing single base substitutions from A to G at positions 957, 993, and 1810 of LGR8. Among the three changes, only the 1810 A to G substitution is associated with an amino acid change from isoleucine to valine (Ile604Val) located in the fifth transmembrane domain of this seven-transmembrane receptor. This change was more frequent in a control group of normal fertile adult males and infant boys than in the group of cryptorchid males. The change is not associated with altered receptor signalling, thus suggesting the presence of a polymorphism unrelated to the cryptorchid phenotype. These data indicate that mutations involving the human LGR8 gene do not represent a frequent cause of cryptorchidism in the Finnish population.

Tumor Necrosis Factor Regulation of Apoptosis in Mouse Preimplantation Embryos and Its Antagonism by Transforming Growth Factor Alpha/Phosphatidylionsitol 3-Kinase Signaling System

Abstract Survival and apoptosis of cells in preimplantation embryos are fundamental for successful pregnancy. Relevant to these processes, tumor necrosis factor (TNF) and transforming growth factor alpha (TGFA) are produced by mammalian oviducts and uteri. In early embryos, TNF induces apoptosis, whereas TGFA could act as a survival factor. Here we investigated the TNF regulation of apoptosis in early mouse embryos and its antagonism by TGFA. TNF receptor superfamily, member 1a mRNA was detectable throughout early embryonic stages, with an increase after the early blastocyst stage, whereas the expression of TNF receptor superfamily, member 1b transcripts were detected only at the expanded blastocyst stage. Although pregnant uteri produced TNF, physiologic levels were low during the preimplantation period. Treatment with TNF inhibited the development of two-cell stage embryos to blastocysts showing decreased proliferation and increased apoptosis both in vitro and in vivo. These detrimental effects of TNF on early embryo development and survival were blocked by a neutralizing anti-TNF antibody. In addition to the death receptor-mediated pathway, TNF-induced apoptosis was further mediated by disruption of mitochondrial functions, characterized by release of cytochrome c and activation of caspase 9. The proapoptotic effects of TNF in blastocysts were counteracted by cotreatment with TGFA. The antagonistic effect of TGFA on TNF-induced apoptosis was blocked by phosphatidylionsitol 3-kinase (PI3K) inhibitors. The present findings demonstrate the stage-selective susceptibility to the apoptosis-inducing effect of TNF in mouse preimplantation embryos and that the TGFA/PI3K signaling system has an important role in the control of TNF-induced apoptosis in blastocysts.

Spontaneous conception after the birth of infants conceived through in vitro fertilization treatment

OBJECTIVE To investigate the incidence of spontaneous conception after the birth of infants conceived through IVF in couples with subfertility caused by endometriosis, a mild male factor, or unexplained factors, and to identify clinical conditions related to the occurrence of spontaneous conception. DESIGN Retrospective analysis. SETTING Department of Obstetrics and Gynecology, Akita University School of Medicine, Akita, Japan. PATIENT(S) One hundred forty-two women who conceived through IVF and 25 of the 142 women who subsequently conceived spontaneously. INTERVENTION(S) Patient characteristics and IVF data were obtained from hospital records, and follow-up data were collected through telephone interviews. MAIN OUTCOME MEASURE(S) Cumulative conception rate. RESULT(S) The cumulative conception rate at 60 months after the delivery of infants conceived through IVF was 18%, with most conceptions occurring within 2 years of delivery. Proportional hazard analysis indicated that patient age was the most important clinical variable related to the occurrence of spontaneous conception. CONCLUSION(S) This study provides information that will be useful in counseling subfertile couples who have conceived through the use of IVF.

First‐trimester diagnosis of conjoined twins after in‐vitro fertilization–embryo transfer (IVF–ET) at blastocyst stage

on postnatal ultrasound and typical characteristics displayed by MRI permitted the diagnosis of hemangioma. These tumors most commonly occur as polypoid masses adherent to the right atrial free walls or pericardium. They tend to be homogeneous, circumscribed, slightly more echogenic than myocardium and sometimes mixed with microcystic spaces. In most cases the vascular nature of the tumor is not demonstrable with color flow mapping as the abnormal vessels are microscopic. However, the main feeding vessel may readily be demonstrated as was seen in the present case. Differential diagnoses include other histological types of cardiac tumor such as rhabdomyoma, fibroma and teratoma. However, prenatal differentiation of hemangioma from these cardiac tumors may often be impossible and computed tomography scan or MRI may be useful for establishing the diagnosis2. Unlike cardiac rhabdomyoma, which regress postnatally, either partially or completely, cardiac hemangioma usually do not regress and usually require surgical removal. The present case was unusual in that the mass gradually regressed, resulting in complete resolution at 6 months.

Studies on Soluble Ectodomain Proteins of Relaxin (LGR7) and Insulin 3 (LGR8) Receptors

Abstract: The ectodomains of both the relaxin (LGR7) and the INSL3 (LGR8) receptors can be expressed on the cell surface using only a single transmembrane domain. These membrane‐anchored proteins retain the ability to bind relaxin and can be cleaved from the cell surface. The subsequent LGR7 protein, 7BP, binds relaxin and can act as a functional relaxin antagonist. By contrast, the equivalent LGR8 protein 8BP does not bind relaxin or antagonize LGR8 activity. The 7BP protein has been successfully immobilized onto chemically derivatized surfaces for the capture of relaxin peptides and subsequent identification via SELDI‐MS analysis.